The pathogenic fungus, Histoplasma capsulatum, is endemic to the Midwestern and Southeastern US and is the most frequent cause of respiratory fungal infection. The organism thrives within the intracellular environ- ment of monocytes (Mo) and macrophages (M?) and has the capacity to establish a latent state. Employing a multidisciplinary approach including metallomics, immunology, cell biology and bioinformatics, our studies have identified novel functions of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 that help to explain their disparate effects on the growth of the fungus with Mo/M?. GM-CSF deprives the organism of zinc intracellularly while concurrently boosting production of reactive oxygen species. The end result is killing of yeast cells. On the other hand, IL-4 promotes intracellular survival by fortifying the amount of zinc available to Histoplasma. Crucial to the activities of GM-CSF and IL-4 are metallothioneins (MTs), which store and donate zinc, and zinc transporters. They are a central node mediating the link between cytokine activation and effector function. For GM-CSF, MTs1 & 2 are essential whereas for IL-4, it is MT3. In this proposal, we will build on our work conducted during the last funding cycle and explore in depth how the MT- zinc axis regulates the activity of GM-CSF and IL-4 on human and mouse Mo/M?. We have collected exciting data that glycolysis governs the expression of MTs1&2 in infected GM-CSF-stimulated Mo/M?. On the other hand, MT3 tempers the glycolytic response in IL-4 activated in these cells. Aim 1 will decipher how glycolysis molds MT1&2 expression and intracellular Zn2+ distribution to bolster Mo/M? effector function. Aim 2 will define the intracellular alterations in GM-CSF-activated Mo/M? that deny the fungus access to zinc and the in vivo effect of MTs1&2 on host defenses exerted by these phagocytes. Aim 3 will elucidate the intersection of glycolysis and MT3 in shaping the physiology of IL-4 on Histoplasma-infected Mo/M?. These studies will provide new insights into cytokine regulation of metabolism and metallobiology in Histoplasma-infected Mo/M?.